* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Poster_02_NN.
Neonatal infection wikipedia , lookup
West Nile fever wikipedia , lookup
Hospital-acquired infection wikipedia , lookup
Onchocerciasis wikipedia , lookup
Cryptosporidiosis wikipedia , lookup
Hepatitis C wikipedia , lookup
Leptospirosis wikipedia , lookup
Plasmodium falciparum wikipedia , lookup
Schistosoma mansoni wikipedia , lookup
Schistosomiasis wikipedia , lookup
Human cytomegalovirus wikipedia , lookup
Hepatitis B wikipedia , lookup
Diagnosis of HIV/AIDS wikipedia , lookup
Middle East respiratory syndrome wikipedia , lookup
Trichinosis wikipedia , lookup
African trypanosomiasis wikipedia , lookup
Toxocariasis wikipedia , lookup
Sarcocystis wikipedia , lookup
Oesophagostomum wikipedia , lookup
Dirofilaria immitis wikipedia , lookup
Use of ELISA to test seroprevalence of Toxoplasma gondii in cats of Tom Green County Amanda Weaver and Dr. Nicholas Negovetich, Department of Biology Introduction Methods Toxoplasma gondii is a protozoan parasite that infects various mammals and birds. This parasite is considered zoonotic because it can be transferred from animals to humans through contact with infected feces or eating undercooked infected meat. When cat feces containing oocysts are accidentally ingested by other animals, the parasite encysts in the muscle of the new host, but will not be able to replicate, enter the environment, and infect other animals. Therefore, cats are essential in the life cycle of T. gondii because they allow this parasite to spread to other animals in the absence of a direct trophic interaction. For this reason, I chose to use cats as a model to measure the prevalence of this parasite in Tom Green County. I collected samples from four veterinary offices in Tom Green County. These veterinarians donated aliquots of blood or serum from samples they took throughout the week for clinical procedures and included the gender and age of the animal from which the sample came. I picked the samples up at the end of each week, kept track of which facility they came from (which will allow me to compare the prevalence from different locations within the county) and kept them in the parasitology lab freezer until I had 90 samples to run the ELISA test. There are several ways to test for T. gondii including serological tests (such as an ELISA), PCR, histological examination, or isolation of the parasite10. I chose to run an ELISA to test my samples because it is economic, makes use of available resources (leftover blood samples), and has 96 wells in each plate so I can run many samples at one time. Since the ELISA tested for IgG antibodies, it detected if the cat has ever been infected with or exposed to T. gondii, and developed antibodies against it. Serum samples were considered positive if the absorbance reading at 450 nm equaled or exceeded 2.1 times the absorbance reading of the negative control. My independent variables of interest include sex (male vs. female), age (divided into separate age groups), and veterinary clinic. Collect Blood Samples Results Run ELISA Statistical Analysis Compare to Literature Figure 5. Graph showing seroprevalence of 4 different clinics with 95% bootstrapped confidence intervals. P-value= 0.23 Figure 1. Life cycle of Toxoplasma gondii. Toxoplasma gondii is a threat to many animals including humans as it causes fever, confusion, headache, seizures, nausea, and poor coordination in immunosuppressed individuals. The CDC approximates 22.5% of people in the United States have been infected, and that number increases to as much as 95% in some parts of the world2. Although infection with T. gondii usually leads to minimal, if any, symptoms, it is thought to cause changes in human behavior1. For example, T. gondii is known to manipulate the behavior of mice to become less fearful of cats which makes them easier for the cats to consume and helps the parasite move forward in its life cycle. Webster et al. claim that T. gondii may also affect human behavior by making us more likely to take risks, i.e., receiving speeding tickets and being involved in work place accidents1. Also, the presence of antibodies to T. gondii were linked to several cases of schizophrenia, among other neurological symptoms1. This parasite is most dangerous, though, when a mother passes it to her child. Congenital toxoplasmosis occurs when women become infected with T. gondii during their pregnancy, with earlier infection causing more severe symptoms for the baby including hydrocephalus, mental retardation, and intracerebral calcifications2. For this reason, it is recommended for a pregnant women to avoid cleaning litter boxes and to wash their hands thoroughly after picking vegetables from a garden because cat feces may be present in the soil. Also, meat should be cooked thoroughly to kill parasites that have encysted in muscle tissue. Discussion Before we ran the ELISA, we were expecting 30%-60%3-9 of our samples to be positive with possibly an even lower number due to the dry conditions of west Texas, which make it less suitable for oocyst survival6. Also, we were expecting older cats to have a higher prevalence than younger cats because they have had more time to be exposed to the parasite5. However, neither one of our expectations were met which were interesting findings for us. We believe the higher than expected prevalence could have been caused by several factors that have had an impact on the parasite’s life cycle including more outdoor than indoor cats, many intermediate hosts, unforeseen helpful conditions in our environment for the oocyst to survive such as people watering their yards, and a warmer winter than usual. The lack of a significance of the prevalence among age groups can be explained by a lack of variance since 90% of the cats were positive. Future Directions: If I were able to continue this study in cats, I would want to sample more cats in our county throughout different times of the year, expand the study to various parts of Texas, and test for IgM antibodies as well as IgG to show how long the cat has been infected. Even though I doubt I will be able to continue this study in cats, I may be able to perform a study on T. gondii in humans while I am in medical school or sometime in the future. ` Figure 6. Graph showing seroprevalence of males and Figure 7. Graph showing seroprevalence of five females with 95% bootstrapped confidence intervals. different age groups with 95% bootstrapped confidence intervals. P-value= 0.37 P-value= 0.35 Acknowledgements Figure 2. ELISA kit including wash solution, enzyme conjugate, substrates, and stop solution. Figure 3. Using micropipette to add serum to wells. Results We found 90% (81/90) of our samples to be positive for IgG antibodies to T. gondii. Once we received our data, we ran statistical analysis using R (www.r-project.org) to determine if sex, age, or clinic had an effect on the seroprevalence of the cats we tested for this parasite. We didn’t find any significant P-values which is a result of a lack of variance since we had 90% positive. We also ran an age-sex interaction term to determine if these two variables combined affected the seroprevalence, but we obtained a P-value of 0.3 which suggests they do not affect seroprevalence. We would like to thank the Angelo State University undergraduate faculty mentored and Head of the River Ranch research grants for helping fund our research. We are also thankful for the veterinary offices (College Hills, Los Caballos, San Angelo, and River Oaks) that collected blood samples and data for our project. Literature Cited 1. Webster, J.P., Kaushik, M., Bristow, G.C., and McConkey, G.A. “Toxoplasma gondii infection, from predation to schizophrenia: can animal behavior help us understand human behavior?” Journal of Experimental Biology. 216, 99-112. 2013. 2. Center for Disease Control and Prevention. “Parasites- Toxoplasma infection.” http://www.cdc.gov/parasites/toxoplasmosis/disease.html. 3. Vollaire, M.R., Radecki, S.V., and Lappin, M.R. “Seroprevalence of Toxoplasma gondii antibodies in clinically ill cats in the United States.” American Journal of Veterinary Research. 66, 874-877. 4. Nutter, F.B., Dubey, J.P., Levine, J.F., Breitshwerdt, E.B., Ford, R.B., and Stoskopf, M.K. “Seroprevalences of antibodies against Bartonella hensalae and Toxoplasma gondii and fecal shedding of Cryptosporidium spp, Giardia spp, and Toxocara cati in feral and pet domestic cats.” Journal of American Veterinary Medical Association. 225, 1394-1398. 5. Dubey, J.P., Bhatia, C.R., Lappin, M.R., Ferreira, L.R., Thorn, A., and Kwok, O.C.H. “Seroprevalence of Toxoplasma gondii and Bartonella spp. Antibodies in cats from Pennsylvania.” Journal of Parasitology. 95, 578-580. 2009. 6. Elmore, S.A., Jones, J.L., Conrad, P.A., Patton, S., Lindsay, D.S., and Dubey, J.P. “Toxoplasma gondii: epidemiology, feline clinical aspects, and prevention.” Cell Press. 26, 190-196. April 2010. 7. Al-Kappany, Y.M., Rajendran, C., Ferreira, L.R., Kwok, O.C.H, Abu-Elwafa, S.A., Hilali, M., and Dubey, J.P. “High prevalence of Toxoplasmosis in cats from Egypt: isolation of viable Toxoplasma gondii, tissue distribution, and isolate designation. Journal of Parasitology. 96, 1115-1118. 2010. 8. Haddadzadeh, H.R., Khazraiinia, P., Aslani, M., Rezaeian, M., Jamshidi, S., Taheri, M., and Bahonar, A. “Seroprevalence of Toxoplasma gondii in stray and household cats in Tehran.” Veterinary Parasitology. 138, 211-216. 9. Castillo-Morales, V.J., Viana, K.Y.A., Guzman-Marin, E.S., Jimenez-Coello, M., Segura-Correa, J.C., Aguilar-Caballero, A.J., and Ortego-Pacheco, A. “Prevalence and Risk Factors of Toxoplasma gondii Infection in Domestic Cats from the Tropics of Mexico Using Serological and Molecular Tests.” Interdisciplinary Perspectives on Infectious Diseases. 2012, Article ID 529108. Figure 4. Picture of the 96 well plate after our ELISA was complete. Yellow is positive and clear is negative. 10. Montoya, J.G. “Laboratory Diagnosis of Toxoplasma gondii Infection and Toxoplasmosis.” Journal of Infectious Diseases. 185, 573-582. 2002.